Research ArticleCELL BIOLOGY

An NF-κB–driven lncRNA orchestrates colitis and circadian clock

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Science Advances  14 Oct 2020:
Vol. 6, no. 42, eabb5202
DOI: 10.1126/sciadv.abb5202
  • Fig. 1 Lnc-UC is a cycling and colitis-related lncRNA.

    (A) Heat map for cycling lncRNAs in colons of colitis and normal mice at six circadian time points. Red indicates high expression, and blue indicates low expression of lncRNAs as shown in the scale bar. (B) Venn diagram showing numbers of cycling, DE (differentially expressed), and CDE (cycling and differentially expressed) lncRNAs. (C) Heat map for CDE lncRNAs in colons of normal mice at six circadian time points. (D) Quantitative polymerase chain reaction measurements of colonic Lnc-UC at six circadian time points. Data are mean ± SD (n = 3). *P < 0.05 at individual time points as determined by two-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. (E) CISH analysis of Lnc-UC expression in colon of WT mice. Scale bar, 50 μm. (F) Lnc-UC levels in PMs and various tissues of WT mice. Data are mean ± SD (n = 5). (G) Cytoplasmic and nuclear levels of Lnc-UC in colon and BMDMs derived from WT mice. Data are mean ± SD (n = 5). (H) FISH analysis of subcellular Lnc-UC (green) location in BMDMs. Scale bar, 10 μm.

  • Fig. 2 NF-κB drives expression of Lnc-UC.

    (A) KEGG pathway analysis of differentially expressed mRNAs associated with colitis at six circadian time points. (B) Heat map for colonic transcripts of NF-κB target genes in colitis and normal mice at six circadian time points. (C) Lnc-UC levels in BMDMs treated with LPS or vehicle for 24 hours. (D) Lnc-UC levels in BMDMs treated with LPS or vehicle for 6, 12, or 24 hours. (E) Lnc-UC levels in serum-shocked BMDMs treated with LPS or vehicle. (F) Lnc-UC levels in PMs treated with LPS or vehicle for 24 hours. (G) FISH analysis of Lnc-UC (green) in BMDMs treated with LPS or vehicle. Scale bar, 10 μm. (H) Lnc-UC levels in BMDMs treated with Tnfα or p65. (I) Lnc-UC and IL-1β levels in BMDMs treated with LPS or JSH-23. (J) Effects of p65 on different versions of Lnc-UC promoters. Data are mean ± SD (n = 6). “X” mark denotes a mutation. (K) ChIP assays showing enrichment of p65 to two κB sites of Lnc-UC promoter (left) and enhanced enrichment by Tnfα (right). A κB site of mCSF (macrophage colony-stimulating factor) promoter and a nonspecific region (NR) of Lnc-UC promoter were used as a positive and negative control, respectively. In (C) to (E), (F), (H), (I), and (K), data are mean ± SD (n = 5). In (C), (F), (H), (J), and (K), *P < 0.05 as determined by Student’s t test; in (D) and (I), *P < 0.05 as determined by one-way ANOVA followed by Bonferroni post hoc test; in (E), *P < 0.05 at individual time points as determined by two-way ANOVA followed by Bonferroni post hoc test.

  • Fig. 3 Lnc-UC acts as an inflammatory regulator.

    (A) KEGG analysis of Lnc-UC–induced differentially expressed genes in BMDMs (NF-κB–related pathways are shown). (B) Gene set enrichment analysis (GSEA) analysis showing activation of NF-κB signaling pathway by Lnc-UC. (C) Lnc-UC overexpression down-regulates NF-κB target genes in LPS-treated BMDMs. (D) Lnc-UC knockdown by ASOs up-regulates NF-κB target genes in LPS-treated BMDMs. (E) Rescue experiments showing that Lnc-UC knockdown–induced changes in expressions of NF-κB target genes can be restored by Lnc-UC overexpression. (F) Effects of Lnc-UC and Bay 11-7082 (Bay) on NF-κB–dependent reporter activity. (G) Effects of Lnc-UC and Bay on the binding of NF-κB DNA probe to nuclear proteins derived from BMDMs. (H) Weight loss measurements of Lnc-UC−/− and WT mice treated with DSS. (I) DAI scores of Lnc-UC−/− and WT mice. (J) Colon lengths of Lnc-UC−/− and WT mice. Photo credit: Y. Lin, College of Pharmacy, Jinan University. (K) MPO activities of Lnc-UC−/− and WT mice. (L) Representative images of hematoxylin and eosin staining in the colon. Scale bars, 100 μm. (M) Histopathological scores of Lnc-UC−/− and WT mice. Survival rate (N) and survival time (O) of WT mice after oxazolone treatment. (P) Survival time of Lnc-UC−/− mice after oxazolone treatment. In (C) to (F), data are mean ± SD (n = 5). In (H) to (K), (M), (O), and (P), data are mean ± SD (n = 8). In (C), (J), (K), and (M), *P < 0.05 as determined by Student’s t test; in (D) to (F), *P < 0.05 as determined by one-way ANOVA followed by Bonferroni post hoc test; in (H) and (I), *P < 0.05 at individual days as determined by two-way ANOVA followed by Bonferroni post hoc test; in (N), *P < 0.05 as determined by log-rank test.

  • Fig. 4 Lnc-UC regulates circadian clock gene Rev-erbα and inflammations.

    (A) Expression of Lnc-UC and Rev-erbα mRNA in the colons of Lnc-UC−/− and WT mice at six circadian time points. (B) Protein expression of Rev-erbα in the colons of Lnc-UC−/− and WT mice at six circadian time points. (C) KEGG analysis of Rev-erbα–induced differentially expressed genes in BMDMs. (D) Rev-erbα ablation up-regulates NF-κB target genes in BMDMs. (E) Venn diagram showing an extensive overlap of Lnc-UC– and Rev-erbα–associated pathways. (F) mRNA levels of Nlrp3 in BMDMs derived from Rev-erbα−/− and WT mice. (G) RIP assays showing no interaction between Rev-erbα protein and Lnc-UC. (H) Effects of Lnc-UC overexpression on mRNA levels of Rev-erbα and Bmal1 in PMs and BMDMs. (I) Effects of Lnc-UC overexpression on protein levels of Rev-erbα and Bmal1 in PMs and BMDMs. (J) Effects of Lnc-UC knockdown by ASO1 and ASO2 on mRNA levels of Rev-erbα and Bmal1 in PMs and BMDMs. In (H) to (J), cells were transfected with overexpression plasmid or ASO for 24 hours before harvest. Data are mean ± SD (n = 5). In (A), *P < 0.05 at individual time points as determined by two-way ANOVA followed by Bonferroni post hoc test; in (D) and (H), *P < 0.05 as determined by Student’s t test; in (F) and (J), *P < 0.05 as determined by one-way ANOVA followed by Bonferroni post hoc test.

  • Fig. 5 Lnc-UC interacts with Cbx1 and modulates Rev-erbα transcription by reducing trimethylation of H3K9.

    (A) Secondary structure of Lnc-UC with minimum free energy predicted by Vienna RNA web server. (B) Silver staining of protein mixtures following an RNA pull-down assay. (C) Western blotting analysis of interactions between Cbx1 protein and different versions of Lnc-UC probes following an RNA pull-down assay. Full-length Lnc-UC (FL), antisense (AS), and truncated fragments are labeled in the top panel. (D) RIP assay showing an interaction between Cbx1 and Lnc-UC. (E) FISH-immunofluorescence assays showing colocalization of Lnc-UC with Cbx1 protein in BMDMs. Nuclei are stained with DAPI (blue). Scale bar, 10 μm. (F) Effects of Lnc-UC mutation on mRNA levels of inflammatory factors in BMDMs. Lnc-UC mut-1/2 [Lnc-UC with mutated sites of (1–74 nt) and (326–432 nt)]. (G) Effects of Lnc-UC knockdown by ASO1 on protein levels of H3K9me3, Rev-erbα, and Gapdh in BMDMs. (H) Lnc-UC knockdown by ASO1 increases enrichments of Cbx1, H3K9me3, and Suv39h1 onto Rev-erbα promoter in BMDMs. (I) Cbx1 knockdown by small interfering RNA (siRNA) decreases enrichment of H3K9me3 onto Rev-erbα promoter in BMDMs. (J) Effects of Cbx1 knockdown on mRNA levels of inflammatory factors in BMDMs. (K) Schematic diagram showing the molecular mechanism for Lnc-UC regulation of Rev-erbα expression. Data are mean ± SD (n = 5). In (D) and (J), *P < 0.05 as determined by Student’s t test; in (H) and (I), *P < 0.05 as determined by two-way ANOVA followed by Bonferroni post hoc test.

  • Fig. 6 Human Lnc-UC (hLnc-UC) is a cycling and anti-inflammatory lncRNA.

    (A) Genomic locations of Lnc-UC and hLnc-UC. (B) CISH analysis of hLnc-UC in human colons. Scale bar, 50 μm. (C) hLnc-UC, Nlrp3, and IL-1β levels in the mucosae of patients with colitis and healthy individuals. (D) Rhythmic expressions of hLnc-UC, BMAL1, REV-ERBα, and NLRP3 in serum-shocked THP-1 cells. (E) hLnc-UC overexpression induces mRNA expression of REV-ERBα and represses inflammatory factors in THP-1 cells. (F) hLnc-UC overexpression increases REV-ERBα protein and reduces NLRP3, phosphorylated p65 (p-p65), and pro–IL-1β in THP-1 cells. (G) hLnc-UC reduces IL-1β and IL-18 in BMDM incubation medium. (H) Rhythmic expression of hLnc-UC in serum-shocked MDMs. (I) hLnc-UC overexpression induces mRNA expression of REV-ERBα and represses inflammatory factors in MDMs. Data are mean ± SD (n = 5). In (C), (E, left) and (I), *P < 0.05 as determined by Student’s t test; in (E, right) and (G), *P < 0.05 as determined by one-way ANOVA followed by Bonferroni post hoc test.

Supplementary Materials

  • Supplementary Materials

    An NF-κB–driven lncRNA orchestrates colitis and circadian clock

    Shuai Wang, Yanke Lin, Feng Li, Zifei Qin, Ziyue Zhou, Lu Gao, Zemin Yang, Zhigang Wang, Baojian Wu

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